| Metal ions are ubiquitous in biological systems and play essential roles in protein structure, gene regulation, electron-transfer, and redox reactions. They can also be toxic to cells if present at high concentration. Bacteria have developed multiple strategies to maintain metal homeostasis. One strategy is to utilize metalloregulatory proteins to sense the intracellular metal ion concentration, and to switch on or off the expression of genes for metal transport. In this work, I studied manganese regulation in Bacillus subtilis.; The Bacillus subtilis yghN gene encodes a protein distantly related to the Corynebacterium diptheriae diptheria toxin repressor (DtxR). While DtxR mediates the iron-dependent repression of iron uptake, I demonstrated that yghN (herein renamed mntR) encodes a manganese-modulated regulator of manganese transport. An mntR mutant strain is sensitive to both manganese and cadmium, suggesting that the transport of these metals is derepressed. By genetic and biochemical methods, I identified two genes that are regulated by MntR. One is mntH and encodes a member of the NRAMP family of proton-coupled, metal ion transporters; and the other is mntABCD and encodes a Mn(II) ABC transporter. MntR represses the transcription of mntH and mntA at high concentrations of Mn(II). Using direct measurements of mRNA levels, I demonstrated that MntR functions as a manganese-responsive transcriptional repressor. Purified MntR protein binds to a ∼55 by region overlapping the mntH transcription starting site. Binding to the mntABCD regulatory region also involves an extended binding interaction: five distinct complexes were observed that lead to DNaseI protection over a region of 130 bp. The sequence requirements for MntR binding in this region were investigated using 5'- and 3'-deletion analyses and point mutations. Mutations in the putative MntR boxes decrease the efficiency of repression without greatly reducing promoter strength but overall, none of the individual sites are essential for binding and repression. This suggests that MntR binds cooperatively to the mntABCD control region.; To explore the global regulation of manganese homeostasis, I combined computer-assisted consensus search with cDNA microarray analysis. More than 600 genes were found to be down-regulated or up regulated in an mntR mutant with or without manganese stress. Strikingly, the PerR regulon and Fur regulon are both repressed by high concentrations of manganese. |
